2.4. Method Sections
3.1.1 Results
3.1.1.4. Inactivation of EGFR and Wnt/β-catenin Signaling Induces Mesenchymal-Epithelial Transition (MET): In the EMT process, tumor-
[Results and Discussions]
3.1.1.4. Inactivation of EGFR and Wnt/β-catenin Signaling Induces
[Results and Discussions]
Figure 3.1.24: Graphical representation of changes in gene expression levels obtained from MDA-MB-231 monolayer cultures following inhibitor treatment quantified by qRT-PCR analysis.
[Results and Discussions]
Figure 3.1.25: Representative Western blots showing E-cadherin, N- cadherin and Vimentin levels in cellular extracts of MDA-MB-231 monolayer cultures. For monolayer cultures, cells were treated with inhibitors for 48 h, where the concentration of lapatinib is 6 µM and of XAV939 is 50 µM. β-actin serves as a loading control. Graphs represent the changes in the expression levels of E- cadherin, N-cadherin and Vimentin with respect to untreated samples following inhibitor treatment. The expression levels were deduced from the blots using ImageJ software.
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[Results and Discussions]
Besides EMT markers, some important proteins and transcription factors, such as Ki-67, Twist and SNAI2, are also implicated in the process of EMT.
Although no significant alterations were observed for the transcription factors Twist and SNAI2, EpCAM and Ki-67 were found to be downregulated by 3.3-fold and 2.9-fold after combination therapy, respectively (Figure. 3.1.26 and Figure.
3.1.27).
The combination effect was also examined in tumor spheroids by qRT-PCR.
Unlike in the monolayer cells, vimentin expression was low, and E-cadherin expression was only marginally elevated, even after 72 h of treatment (Figure.
3.1.28). However, other EMT markers such as N-cadherin and fibronectin were not found to be significantly altered after combination treatment (Figure. 3.1.28).
While combination treatment led to decreased Twist1 expression, lapatinib alone increased its expression by two-fold. SNAI2 was upregulated under both treatment conditions (Figure. 3.1.29). Exogenous EpCAM expression in TNBC cells has been found to promote EMT, to induce a CSC-like phenotype and to enhance metastasis in vitro and in vivo [100]. It was found that after combination treatment, EpCAM was downregulated by 3.30-fold in monolayers but remained unaltered in spheroids (Figure. 3.1.30).
Expression analysis of key EMT related proteins were also carried out in spheroids. Remarkably, a 14.3-fold decrease in vimentin expression was observed in MDA-MB-231 spheroids following co-treatment (Figure. 3.1.31). Expression analysis of EMT-related proteins in two other cell lines, MDA-MB-468 and MCF- 7, yielded similar EMT reversal results (Figure. 3.1.32 and Figure. 3.1.33). E- cadherin was, however, found to be downregulated in MCF-7 cells. The fold change in protein expression of EMT markers are presented in Table. 3.1.4.
[Results and Discussions]
Figure 3.1.26: Graphical representation of changes in gene expression levels obtained from MDA-MB-231 monolayer cultures following inhibitor treatment quantified by qRT-PCR analysis.
Figure 3.1.27: Graphical representation of changes in gene expression levels obtained from MDA-MB-231 monolayer cultures following inhibitor treatment quantified by qRT-PCR analysis.
[Results and Discussions]
Figure 3.1.28: Graphical representation of changes in gene expression levels obtained from MDA-MB-231 spheroids following inhibitor treatment quantified by qRT-PCR analysis.
Figure 3.1.29: Graphical representation of changes in gene expression levels obtained from MDA-MB-231 spheroids following inhibitor treatment quantified by qRT-PCR analysis.
[Results and Discussions]
Figure 3.1.30: Graphical representation of changes in gene expression levels obtained from MDA-MB-231 spheroids following inhibitor treatment quantified by qRT-PCR analysis.
Figure 3.1.31: Representative Western blots showing Vimentin levels in cellular extracts of MDA-MB-231 spheroids. Spheroids were treated using 30 µM lapatinib and 25 µM XAV939 for 72 h. β-actin serves as a loading control. Graphs represent the changes in the expression level Vimentin with respect to untreated samples following inhibitor treatment. The expression levels were deduced from the blots using ImageJ software.
[Results and Discussions]
Figure 3.1.32: Representative Western blots showing (a) E-cadherin and (b) N-cadherin levels in cellular extracts of MDA-MB-468 monolayer cultures. β-actin serves as a loading control. (c) The histogram of immunofluorescence flowcytometry detecting Vimentin of MDA-MB-468 monolayer cultures. Graphs represent the changes in the expression levels of E-cadherin, N-cadherin and Vimentin with respect to untreated samples following inhibitor treatment. The expression levels were deduced from the blots using ImageJ software.
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[Results and Discussions]
Figure 3.1.33: Representative Western blots showing (a) E-cadherin and (b) N-cadherin and Vimentin levels in cellular extracts of MCF-7 monolayer cultures.
β-actin serves as a loading control. Graphs represent the changes in the expression levels of E-cadherin, N-cadherin and Vimentin with respect to untreated samples following inhibitor treatment. The expression levels were deduced from the blots using ImageJ software.
Fold change in protein expression
Protein MDA-MB-231 MDA-MB-468 MCF-7
E-cadherin 1.73 1.78 2.38
N-cadherin 3 3.1 2.1
Vimentin 2.18 2 1.17
Table. 3.1.4: Tabular representation of fold change of EMT markers with respect to untreated control cells following treatment with Lapatinib+XAV939.
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[Results and Discussions]
MDM2 plays a critical role in controlling p53 and, thus, apoptosis. MDM2 acts as a cellular antagonist of p53 [101]. It has also been reported that MDM2 increases drug resistance through inducing EMT independent of p53 [102]. Co- targeting EGFR and Wnt/β-catenin led to a decreased MDM2 level both in monolayers (1.26-fold) and tumor spheroids (4.16-fold), which incurred cell death following co-treatment (Figure. 3.1.34 and Figure. 3.1.35). It is well-known that caveolin-1 overexpression is correlated with migration, metastasis and drug resistance [103]. Co-treatment reduced the caveolin-1 expression level by 1.72-fold and 1.50-fold in monolayers and spheroids, respectively (Figure. 3.1.34 and Figure.
3.1.35). Together, these findings indicate that combined treatment with EGFR and Wnt/β catenin signaling inhibitors induces MET and concomitant reductions in the metastatic and invasive potentials of TNBC cells.
[Results and Discussions]
Figure 3.1.34: Graphical representation of changes in gene expression levels obtained from MDA-MB-231 monolayer culture following inhibitor treatment quantified by qRT-PCR analysis.
Figure 3.1.35: Graphical representation of changes in gene expression levels obtained from MDA-MB-231 spheroids following inhibitor treatment quantified by qRT-PCR analysis.
[Results and Discussions]
3.1.1.5. Co-targeting EGFR and Wnt/β-catenin Signaling Decreases TNBC